The Bradford Protein Assay

The Bradford Assay is a form of colorimetric and spectroscopic analysis developed to determine the concentration of a protein; in an aqueous solution. Produced by Marion Bradford in 1976, it was an innovation of its time due to various factors including its simplicity, fast results, reproducibility and a high sensitivity of 0−0.01 mg (Martina and Vojtech, 2015); in comparison to other assays such as the Lowry and Biuret method, which it succeeded.

The Bradford Reagent is made up of a mixture of Coomassie Brilliant Blue G-250 dye dissolved in a mixture of phosphoric acid and methanol. The assay is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its blue form as it binds to the protein being assayed.

The binding between the dye and the protein occurs as a result of interactions between basic amino acids residues on the proteins and the lone pair of electrons from the dye. This causes the protein’s native shape to be distorted and some of its hydrophobic residues to be exposed. These hydrophobic residues bind to non-polar regions of the dye resulting in van der Waals forces. Furthermore, these van der Waals forces lead to the positioning of the positive amine groups closer to the negative charge of the dye which allows the protein -dye complex to be reinforced through the ionic binding. This results in the colour of the dye from red to blue which we can detect best at 595 nm wavelength of light.

Overall this shows that, the instability of the Coomassie Brilliant Blue G-250 dye is stabilized by binding to the protein. Therefore, as protein content increases, more protein-dye complexes are formed, and more colouration occurs which can be; thus the amount of the complex present in solution is a measure for the protein concentration, and can be estimated by use of an absorbance reading.

As there is a direct relationship between the absorbance of a solution and the concentration of soluble protein. This is known according to the Beer-Lambert law, which states that the concentration of a solute is proportional to the absorbance.

The objectives of the experiment were as follows:

• To create a dilution series of protein standard with bovine serum albumin (BSA) and carry out the Bradford protein assay on these known protein concentrations.
• To use the preceding concentrations to create a calibration graph measured against absorbance and use this graph to find out the concentration in protein in two cell extract samples.
• Overall, the experiment was conducted to determine the concentration of protein in two cell extracts of unknown protein concentration using the Bradford protein assay.